Immunogenicity can be significantly improved if an antigen is co-administered with an adjuvant, commonly used as 0.001% to 50% solution in phosphate buffered saline (PBS). Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
Immunostimulatory agents or adjuvants have been used for many years to improve the host immune response to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccarides, normally are the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune response. Aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum.
A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes. To efficiently induce humoral immune response (HIR) and cell-mediated immunity (CMI), immunogens are preferably emulsified in adjuvants.
Desirable characteristics of ideal adjuvants include any or all of:                (1) lack of toxicity;        (2) ability to stimulate a long-lasting immune response;        (3) simplicity of manufacture and stability in long-term storage;        (4) ability to elicit both CMI and HIR to antigens administered by various routes;        (5) synergy with other adjuvants;        (6) capability of selectively interacting with populations of antigen presenting cells (APC);        (7) ability to specifically elicit appropriate TH1 or TH2 cell-specific immune responses; and        (8) ability to selectively increase appropriate antibody isotype levels (for example IgA) against antigens.        
U.S. Pat. No. 4,855,283 granted to Lockhoff et al. on Aug. 8, 1989 which is incorporated herein by reference thereto teaches glycolipid analogs including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immune-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Pat. No. 4,855,283) reported that N-glycolipids analogs displaying structural similarities to the naturally occurring glycolipids, such as glycosphingolipids and glycoglycerolipids, are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudorabies virus vaccine. Some glycolipids have been synthesized from long chain alkylamines and fatty acids that are linked directly with the sugar through the anomeric carbon atom, to mimic the functions of the naturally occurring lipid residues.
U.S. Pat. No. 4,258,029 granted to Moloney, assigned to Connaught Laboratories Limited and incorporated herein by reference thereto, teaches that octadecyl tyrosine hydrochloride (OTH) functions as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and III poliomyelitis virus vaccine. Octodecyl esters of aromatic amino acids complexed with a recombinant hepatitis B surface antigen, enhanced the host immune responses against hepatitis B virus.
Bessler et al., “Synthetic lipopeptides as novel adjuvants,” in the 44th Forum In Immunology (1992) at page 548 et seq., especially at 548–550, incorporated herein by reference, is directed to employing lipopeptides as adjuvants when given in combination with an antigen. The lipopeptides typically had P3C as the lipidated moiety and up to only 5 amino acids, e.g., P3C-SG, P3C-SK4, P3C-SS, P3C-SSNA, P3C-SSNA. The lipopeptide was coupled with or added to only certain antigens or to non-immunogenic proteins, such as P3C-SSNA supplementing S. typhimurium vaccine, PC3-SS coupled to VP1 (135–154) of foot-and-mouth disease, PC3-SG-OSu coupled to non-immunogenic protein hirudin, P3C-SK coupled to FITC or DNP or P3C-SG coupled to a metabolite from Streptomyces venezuelae. While adjuvant mixing and conjugating procedures of Bessler can be employed in the practice of the present invention, Bessler fails to teach or suggest employing a lipoprotein with at least one antigen in a composition, especially such compositions wherein the lipoprotein is also antigenic, or the immunological combination compositions and methods of this invention.
In this regard, a distinction between a peptide, especially a peptide having up to only about 5 amino acids, and a protein is being made, as is a distinction between an antigenic lipoprotein and a non-antigenic lipopeptide, inter alia. Peptides differ immunologically from proteins in that short peptides have the potential for direct presentation by the major histocompatibility complex (MHC), while proteins require processing prior to presentation to T-cells. A peptide further differs from a protein in that a protein is large enough that it is capable of forming functional domains (i.e., having tertiary structure), whereas a peptide cannot.
Nardelli et al. [Vaccine (1994), 12(14):1335–1339] covalently linked a tetravalent multiple antigen peptide containing a gp120 sequence to a lipid moiety and orally administered the resulting synthetic lipopeptide to mice. It was found that both mucosal IgA response and systemic plasma IgG were stimulated, and cell-mediated immunity, as shown by lymphokine production and generation of a specific cytotoxic response, was induced. Only a short peptide was used, rather than a whole lipoprotein, and there is no teaching or suggestion that the synthetic lipopeptide could be used as an adjuvant for other proteins. In fact, this reference actually teaches away from the use of lipoproteins, which are more soluble than lipopeptides, as immunogens; see, e.g. p. 1338, last line (“soluble proteins are not immunogens by oral routes”).
Croft et al. [J. Immunol. (1991), 146(5): 793–796] have covalently coupled integral membrane proteins (Imps) isolated from E. coli to various antigens and obtained enhanced immune responses by intramuscular injection into mice and rabbits. However, there are disadvantages to coupling the lipoprotein and the antigen covalently. Important epitopes may be damaged, and the coupling procedure is difficult to control and often requires the use of toxic cross-linkers. Thus, it would be advantageous to provide a method for inducing an enhanced immunological response which does not require that the antigen be cross-linked to a protein. Moreover, when the antigen CSP-OVA was merely mixed, rather than covalently linked, with the lipoprotein TraT, only a small increase in antibody response was obtained. Croft et al. therefore concluded that the lipid is not necessary for the adjuvant effect, contrary to the surprising findings of the present inventors.
U.S. Pat. No. 4,439,425 relates to lipopeptides having 2 to 10 amino acids and their prophylactic administration by oral or rectal routes.
Bessler et al. [“Synthetic Lipopeptide Conjugates Constitute Efficient Novel Immunogens and Adjuvants in Parenteral and Oral Immunization” (Abstract), Meeting on Molecular Approaches to the Control of Infectious Diseases, (Sep. 13–17, 1995), Cold Spring, Harbor Laboratory (not prior art in view of Jun. 7, 1995 filing date of U.S. Ser. No. 08/476,656)] relates to the oral administration of lipopeptides having six amino acids which were covalently coupled to antigens. The lipopeptide-antigen conjugates were found to induce a hapten-specific immune response.
Schlecht et al. [Zbl. Bakt. (1989) 271:493–500] relates to Salmonella typhimurium vaccines supplemented with synthetically prepared derivatives of a bacterial lipoprotein having five amino acids. The vaccines were administered by two intraperitoneal injections and challenged intraperitoneally with graded doses of S. typhimurium. When the protective capacity of the supplemented vaccines was compared with that of the unsupplemented vaccine, it was found that 90% of the S. typhimurium vaccine could be replaced by the lipopeptide without a recognizable decrease in protective capacity.
Substantial effort has been directed toward the development of a vaccine for Lyme disease. Two distinct approaches have been used for vaccine development. One approach is to use a vaccine composed of whole inactivated spirochetes, as described by Johnson in U.S. Pat. No. 4,721,617. A whole inactivated vaccine has been shown to protect hamsters from challenge and has been licensed for use in dogs.
Due to the concerns about cross-reactive antigens within a whole cell preparation, human vaccine research has focused on the identification and development of non-cross-reactive protective antigens expressed by B. burgdorferi. Several candidate antigens have been identified to date. Much of this effort has focused on the most abundant outer surface protein of B. burgdorferi, namely outer surface protein A (OspA), as described in published PCT patent application WO 92/14488, assigned to the assignee hereof. Several versions of this protein have been shown to induce protective immunity in mouse, hamster and dog challenge studies. Clinical trials in humans have shown the formulations of OspA to be safe and immunogenic in humans [Keller et al., JAMA (1994) 271:1764–1768]. Indeed, one formulation containing recombinant lipidated OspA as described in the aforementioned WO 92/14488, is now undergoing Phase III safety/efficacy trials in humans.
While OspA is expressed in the vast majority of clinical isolates of B. burgdorferi from North America, a different picture has emerged from examination of the clinical Borrelia isolates in Europe. In Europe, Lyme disease is caused by three genospecies of Borrelia, namely B. burgdorferi, B. garinii and B. afzelli. In approximately half of the European isolates, OspA is not the most abundant outer surface protein. A second outer surface protein C (OspC) is the major surface antigen found on these spirochetes. In fact, a number of European clinical isolates that do not express OspA have been identified. Immunization of gerbils and mice with purified recombinant OspC produces protective immunity to B. burgdorferi strains expressing the homologous OspC protein [V. Preac-Mursic et al., INFECTION (1992) 20:342–349; W. S. Probert et al., INFECTION AND IMMUNITY (1994) 62:1920–1926]. The OspC protein is currently being considered as a possible component of a second generation Lyme vaccine formulation.
Recombinant proteins are promising vaccine or immunogenic composition candidates, because they can be produced at high yield and purity and manipulated to maximize desirable activities and minimize undesirable ones. However, because they can be poorly immunogenic, methods to enhance the immune response to recombinant proteins are important in the development of vaccines or immunogenic compositions. Moreover, it would be greatly desired to be able to administer such proteins in combination with other antigens.
A very promising immune stimulator is the lipid moiety N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine, abbreviated Pam3Cys. This moiety is found at the amino terminus of the bacterial lipoproteins which are synthesized with a signal sequence that specifies lipid attachment and cleavage by signal peptidase II. Synthetic peptides that by themselves are not immunogenic induce a strong antibody response when covalently coupled to Pam3Cys [Bessler et al. (1992)].
In addition to an antibody response, one often needs to induce a cellular immune response, particularly cytoxic T lymphocytes (CTLs). Pam3Cys-coupled synthetic peptides are extremely potent inducers of CTLs, but no one has yet reported CTL induction by large recombinant lipoproteins.
The nucleic acid sequence and encoded amino acid sequence for OspA are known for several B. burgdorferi clinical isolates and is described, for example, in published PCT application WO 90/04411 (Symbicom AB) for B31 strain of B. burgdorferi and in Johnson et al., Infect. Immun. 60:1845–1853 for a comparison of the ospA operons of three B. burgdorferi isolates of different geographic origins, namely B31, ACA1 and Ip90.
As described in WO 90/04411, an analysis of the DNA sequence for the B31 strain shows that the OspA is encoded by an open reading frame of 819 nucleotides starting at position 151 of the DNA sequence and terminating at position 970 of the DNA sequence (see FIG. 1 therein). The first sixteen amino acid residues of OspA constitute a hydrophobic signal sequence of OspA. The primary translation product of the full length B. burgdorferi gene contains a hydrophobic N-terminal signal sequence which is a substrate for the attachment of a diacyl glycerol to the sulfhydryl side chain of the adjacent cysteine residue. Following this attachment, cleavage by signal peptidase II and the attachment of a third fatty acid to the N-terminus occurs. The complete lipid moiety is termed Pam3Cys. It has been shown that lipidation of OspA is necessary for immunogenicity, since OspA lipoprotein with an N-terminal Pam3Cys moiety stimulated a strong antibody response, while OspA lacking the attached lipid did not induce any detectable antibodies [Erdile et al., Infect. Immun., (1993), 61:81–90].
Published international patent application WO 91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH) describes the DNA sequence of the ospC gene of B. burgdorferi strain Pko and the OspC (termed pC in this reference) protein encoded thereby of 22 kDa molecular weight. This sequence reveals that OspC is a lipoprotein that employs a signal sequence similar to that used for OspA. Based on the findings regarding OspA, one might expect that lipidation of recombinant OspC would be useful to enhance its immunogenicity; but, as discussed in above-referenced Ser. No. 08/475,781, filed Jun. 7, 1995 (abandoned; the continuation of which is Ser. No. 09/067,453, filed Apr. 28, 1998 (now U.S. Pat. No. 6,538,118 B1)), the therein applicants experienced difficulties in obtaining detectable expression of recombinant OspC. It would be useful to enhance the immunogenicity of recombinant OspC. Moreover, it would be useful to have a multivalent Lyme Disease immunological composition which contains antigens against both North American and European Borrelia isolates.
Streptoccus pneumoniae causes more fatal infections world-wide than almost any other pathogen. In the U.S.A., deaths caused by S. pneumoniae rival in numbers those caused by AIDS. Most fatal pneumoccal infections in the U.S.A. occur in individuals over 65 years of age, in whom S. pneumoniae is the most common cause of community-acquired pneumonia. In the developed world, most pneumococcal deaths occur in the elderly, or in immunodeficient patents including those with sickle cell disease. In the less-developed areas of the world, pneumococcal infection is one of the largest causes of death among children less than 5 years of age. The increase in the frequency of multiple antibiotic resistance among pneumococci and the prohibitive cost of drug treatment in poor countries make the present prospect for control of pneumococcal disease problematical.
The reservoir of pneumococci that infect man is maintained primarily via nasopharyngeal human carriage. Humans acquire pneumococci first through aerosols or by direct contact. Pneumococci first colonize the upper airways and can remain in nasal mucosa for weeks or months. As many as 50% or more of young children and the elderly are colonized. In most cases, this colonization results in no apparent infection. In some individuals, however, the organism carried in the nasopharynx can give rise to symptomatic sinusitis of middle ear infection. If pneumococci are aspirated into the lung, especially with food particles or mucus, they can cause pneumonia. Infections at these sites generally shed some pneumococci into the blood where they can lead to sepsis, especially if they continue to be shed in large numbers from the original focus of infection. Pneumococci in the blood can reach the brain where they can cause meningitis. Although pneumococcal meningitis is less common than other infections caused by these bacteria, it is particularly devastating; some 10% of patients die and greater than 50% of the remainder have life-long neurological sequelae.
In elderly adults, the present 23-valent capsular polysaccharide vaccine is about 60% effective against invasive pneumococcal disease with strains of the capsular types included in the vaccine. The 23-valent vaccine is not effective in children less than 2 years of age because of their inability to make adequate responses to most polysaccharides. Improved vaccines that can protect children and adults against invasive infections with pneumococci would help reduce some of the most deleterious aspects of this disease.
The S. pneumoniae cell surface protein PspA has been demonstrated to be a virulence factor and a protective antigen. In published international patent application WO 92/14488, there are described the DNA sequences for the pspA gene from S. pneumoniae Rx1, the production of a truncated form of PspA by genetic engineering, and the demonstration that such truncated form of PspA confers protection in mice to challenge with live pneumococci.
In an effort to develop a vaccine or immunogenic composition based on PspA, PspA has been recombinantly expressed in E. coli. It has been found that in order to efficiently express PspA, it is useful to truncate the mature PspA molecule of the Rx1 strain from its normal length of 589 amino acids to that of 314 amino acids comprising amino acids 1 to 314. This region of the PspA molecule contains most, if not all, of the protective epitopes of PspA. However, immunogenicity and protection studies in mice have demonstrated that the truncated recombinant form of PspA is not immunogenic in naive mice. Thus, it would be useful to improve the immunogenicity of recombinant PspA and fragments thereof. Moreover, it would be highly desirable to employ a pneumococcal antigen in a combination or multivalent composition. For instance, influenza (Flu) is a problematical infection, especially in the elderly and the young, as well as pneumonia; and, yearly Flu shots are common, especially in North America. Thus, it would be desirable to be able to administer Flu and pneumococcal antigens in one preparation.
Helicobacter pylori is the spiral bacterium which selectively colonizes human gastric mucin-secreting cells and is the causative agent in most cases of nonerosive, gastritis in humans. Recent research activity indicates that H. pylori, which has a high urease activity, is responsible for most peptic ulcers as well as many gastric cancers. Many studies have suggested that urease, a complex of the products of the ureA and ureB genes, may be a protective antigen. However, until now it has not been known how to produce a sufficient mucosal immune response to urease without cholera toxin or related adjuvants.
Antigens or immunogenic fragments thereof stimulate an immune response when administered to a host. Such antigens, especially when recombinantly produced, may elicit a stronger response when administered in conjunction with adjuvant. Currently, alum is the only adjuvant licensed for human use, although hundreds of experimental adjuvants such as cholera toxin B are being tested. However, these adjuvants have deficiencies. For instance, while cholera toxin is a good adjuvant, it is highly toxic. On the other hand, cholera toxin B, while non-toxic, has no adjuvant activity. It would thus be desirable to provide immunological compositions capable of eliciting a strong response without the need for an adjuvant.
In certain instances when multiple antigens (two or more) are administered in the same preparation or sequentially, a phenomenon called efficacy interference occurs. Simply, due to the interaction of one or more antigens in the preparation with the host immunological system, the second or other antigens in the preparation fail to elicit a sufficient response, i.e., the efficacy of the latter antigen(s) is interfered with by the former antigen(s). It would thus be desirable to provide multivalent immunological compositions which do not give rise to this efficacy interference phenomenon; for instance, without wishing to necessarily be bound by any one particular theory, because the second antigen is a lipoprotein and as such is having an adjuvanting effect on the first antigen and, when in a combination composition with an adjuvant, a synergistic potentiating effect is obtained (whereby the first antigen is not interfering with the second antigen and vice versa).
More generally it would be desirable to enhance the immunogenicity of antigens by methods other than the use of an adjuvant, and to have the ability to employ such a means for enhanced immunogenicity with an adjuvant, so as to obtain an even greater immunological response.
Above-referenced U.S. Pat. No. 6,042,838 discloses that mucosal administration of killed whole pneumococci, lysate of pneumococci or isolated and purified PspA, as well as immunogenic fragments thereof, particularly when administered with an adjuvant, provides protection in animals against pneumococcal colonization and systemic infection. It has now been surprisingly found that mucosal administration of other antigens, such as urease, along with a lipoprotein, elicits systemic and local responses in animals without the use of an adjuvant.
It is believed that heretofore the art has not taught or suggested: immunological compositions comprising at least one antigen and a lipoprotein, and, optionally, an adjuvant, more preferably an antigen, an antigenic lipoprotein and, optionally, an adjuvant, and methods for administering the same as a multivalent composition, or for administering those components simultaneously or sequentially, especially such compositions and methods having enhanced immunogenicity.